Pro-Russian separatists have shot down a large military transport plane in eastern Ukraine, reportedly killing 49 people on board in a major blow for the government's efforts to quell the insurgency.

The defence ministry said the plane was shot down as it approached an airport at Luhansk. The Il-76 transport plane had been carrying service personnel, equipment and food. The military spokesman Vladislav Seleznyov told the Reuters news agency that 49 people had died.

The toll would be the biggest suffered by government forces in a single incident since Kiev began a military operation to try to defeat the insurgency in eastern Ukraine.

A defence ministry statement said the rebels "cynically and perfidiously" shot down the plane using anti-aircraft guns and heavy calibre machine guns.

An Il-76 is a four-engined jet that can be used to transport heavy equipment and people. Luhansk, near Ukraine's eastern border with Russia, is an area where separatists have seized government buildings and declared independence after holding disputed referendums.

On Saturday, the Russian energy firm Gazprom said preparations were underway to avert gas supply cuts and possible flow disruption to Europe amid demands for Ukraine to pay £1.15bn of its debts by Monday.

"We are ready to seek compromise, but it is useless to put pressure on us," Gazprom's Sergei Kupriyanov said. Previous talks between Russia and Ukraine, brokered by the European commission, have ended without a firm agreement.

On Friday the US state department said a convoy of military vehicles, including three tanks, had been transported from a Russian depot into Ukraine this week. Three T-64 tanks, multiple rocket launchers and other military vehicles crossed from Russia into Ukraine near the town of Snizhne, the state department said, describing the action as unacceptable. The vehicles – apparently out-of-use Russian tanks – appear to have been commandeered by Ukrainian separatist forces.

Ukraine first raised concerns about the tanks on Thursday, after several reports, including video footage uploaded to YouTube (video), appeared to show them passing through Snizhne.

Kiev maintains that the pro-Russian forces who facilitated Russia's annexation of Crimea and were now laying claim to parts of eastern Ukraine were being covertly supported by Moscow. Russia has denied direct military involvement in eastern Ukraine.

Ukraine's newly elected president, Petro Poroshenko, raised concerns about the tanks directly with his Russian counterpart, Vladimir Putin, on Thursday. Russia has not officially responded to the claims.

On Friday, the US state department deputy spokesperson, Marie Harf, said it believed separatists in eastern Ukraine had "acquired heavy weapons and military equipment from Russia, including Russian tanks and multiple rocket launchers". She said internet videos had shown the same type of tanks that crossed the border in the eastern cities of Snizhne, Torez and Makiyivka.

The statement did not explicitly blame Russia for supplying the weaponry directly to the Ukrainian separatists. But it stated Washington's unequivocal belief that the tanks had crossed the border and hinted at Moscow's complicity. The US secretary of state, John Kerry, had also raised the issue with his Russian counterpart, Sergei Lavrov, according to the state department.

"We have information that Russia has accumulated tanks of a type no longer used by Russian forces at a deployment site in south-west Russia, and some of these tanks recently departed," Harf said.

"Russia will claim these tanks were taken from Ukrainian forces, but no Ukrainian tank units have been operating in that area. We are confident that these tanks came from Russia."

Harf added: "We also have information that Russia has accumulated multiple rocket launchers at this same deployment site in south-west Russia, and these rocket launchers also recently departed."

Earlier on Friday, Harf told reporters: "They [the tanks] were somehow pulled out of the Russian warehouses, someone taught them how to use them, and they were sent from Russia to Ukraine."

Also on Friday, the Associated Press reported that Ukrainian troops had attacked pro-Russia separatists in Mariupol, driving them out of buildings they had occupied in the southern port. Mariupol is the second-largest city in the eastern Donetsk region, where armed separatists have declared independence from the government in Kiev.